C6219

Cx43 staining was accomplished using anti-Cx43 antibodies (C6219, Sigma, 1:2000) and detected using a DAB system (R&D Systems, Minneapolis MN, USA). The images were acquired using a FSX1000 (Olympus, Tokyo, Japan). Quantification of Cx43 present at gap junctions was evaluated by measuring the area of Cx43 at gap junctions and total Cx43 using Image J 1.53a software (NIH, USA).
Mice were perfused with PBS and then fixed overnight in a formalin- and methanol-based fixative solution (Ufix, Sakura Fine Tech, Japan). The following day, the fixed tissues were dehydrated and embedded in paraffin. To quantify cardiac fibrosis, we stained heart sections with Picrosirius red.
Paraffin-embedded fixed heart tissues were cut into 5-µm-thick sections, after which the sections were dewaxed, the antigens were retrieved using microwave antigen retrieval (pH 8.0), and the sections were incubated first with mouse anti-N-cadherin antibody (33-3900, ThermoFisher, USA, 1:500) and rabbit anti-CX43 antibody (C6219, Sigma, USA, 1:2000), and then with Alexa-Fluor-488-conjugated anti-mouse-IgG (A-11001, ThermoFisher, USA, 1:2000) and Alexa-Fluor-635-conjugated anti-rabbit-IgG (A-31576, ThermoFisher, USA, 1:2000). The heart sections were visualized using confocal microscopy (LSM 5100 Meta, Carl Zeiss, Germany).

Normal human bladder tissue was obtained from a 67-year-old female patient who underwent anterior pelvic exenteration under the diagnosis of urethral malignant melanoma after informed consent. Immunostaining with Cx43 antibody (C6219, Sigma-Aldrich, St. Louis, MO, USA) was performed with normal testis as a positive control. Normal mouse bladder tissue was obtained from ten-week-old female C57BL/6 mice. Immunostaining with Cx43 antibody (C6219, Sigma-Aldrich) was performed with normal heart as a positive control. All human experiments were performed in accordance with guidelines on the use of human materials in Kyoto University, and all human experiments were approved by the ethical committees at Kyoto University Hospital. All immunostaining was performed at the Center for Anatomical, Pathological and Forensic Medical Researches in Kyoto University.

Euthanized mice were perfused with 10 ml 1× ice-cold PBS and the hearts were frozen in optimal cutting temperature compound (Fisher Scientific, Ontario, Canada) in dry ice-chilled 2-methylbutane (Sigma, M32631). Cardiac sections were prepared at 5-µm thickness. Standard Mayer’s hematoxylin and eosin (H&E) and Masson’s Trichrome staining were performed by the Toronto Oral Pathology Service (Faculty of Dentistry, University of Toronto, Toronto). Immunofluorescence was performed^{16 (link)} using antibodies and their dilution are listed below. Cx43 (1:100; C6219, Sigma), Cox4 (1:50, ab16056, Abcam), NCAD (1:20 unconcentrated hybridoma supernatant; MNCD2, DSHB), desmin (1:50 unconcentrated hybridoma supernatant; D3, DHSB), Alexa Fluor-568 phalloidin (A12380, Invitrogen, 150 nM), and desmoglein 2 (ab150372, Abcam, 1:250), NaV1.5 (1:500; ASC-005, Alomone Labs), NaV1.5 rabbit sera (1:50, in house⁴ ) and purified β1 (1:500, in house⁴ ).